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AN62502_E 12/07S
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Conclusions
The approach presented here provides a sensitive and
selective method for preparing and analyzing horse plasma
for the presence of rhEPO or DPO. The advantages of this
method include the ability to use up to six diagnostic
peptides to confirm or refute the presence of either illegal
protein-based drug.
The use of stable-isotope labeled analogues provides
further means of confirming the presence of diagnostic
peptides based on chromatographic retention times and
ion ratios.
The sensitivity demonstrated enabled detection up to
72 hours following the last administration of rhEPO,
increasing the confidence that the described method is
useful in the racing industry to maintain a level field of
competition.
Of particular interest is the measured sensitivity that
was achieved using microspray, increasing the analysis
time while simplifying the experimental method and thus,
enabling more laboratories the option of employing
rhEPO/DPO screening.
References
1 Guan, F., Uboh, C., E., Soma, L. R., Birks, E., Li, X., Chen, Y., Mbuy, G.,
LC-MS/MS Method in the Confirmation of Recombinant Human
Erythropoietin and Darbepoietin-alpha in Equine Plasma, accepted by
Analytical Chemistry (see WP 245 for additional applications).
2 Krantz, S. B. Erythorpoietin, Blood , 1991, 77, 419-434.
3 Egrie, J. C., Browne, J. K., Development and Characterization of Novel
Erythropoiesis Stimulating Protein (NESP), British Journal of Cancer,
2001, 84, 3-10.
4 Piercy, R. J., Swardson, C. J., Hinchcliff, K. W., Erythroid Hypoplasia and
Anemia Following Administration of Recombinant Human Erythropoietin
to Two Horses, Journal of the American Veterinary Medical Association,
1998, 212, 244-247.
Acknowledgements
We would like to thank Amgen, Inc (Thousand Oaks, CA) for kindly
donating the rhEPO and DPO standards used in this study, and the PA
Racing Commissions for financial support.
0
1
2
3
4
5
6
7
T4 and T6
ng/ mL of horse plasma
Time Between Administration and Plasma Collection
(hrs)
8,000 International Units Administered (200 IU = 1 ug)
T4
T6
0
0.05
0.1
0.15
0.2
0.25
0.06 ng/mL
0.23 ng/mL
0 10 20 30 40 50 60 70 80
0 20 40 60 80
Figure 7: Calculated rhEPO levels in
extracted horse plasma as a function of
time delay between administration and
sample collection. The calculated levels
were based on the area ratios of T4 and
T6 targeted rhEPO peptides and the
labeled internal standards.
460.300
m/z
573.400
m/z
0
20
40
60
80
100
RelativeAbundance
460.30
573.40
460.300
m/z
573.400
m/z
0
20
40
60
80
100
RelativeAbundance
460.30
573.40
460.300
m/z
573.400
m/z
0
20
40
60
80
100
RelativeAbundance
460.30
573.40
467.300
m/z
580.400
m/z
0
20
40
60
80
100
RelativeAbundance
467.30
580.40
467.300
m/z
580.400
m/z
0
20
60
80
100
RelativeAbundance
40
467.30
580.40
467.300
m/z
580.400
m/z
0
20
40
60
80
100
RelativeAbundance
467.30
580.40
Labeled
rhEPO
10 hrs 72 hrs 0.5 hrs
rhEPO rhEPO rhEPO
Labeled
rhEPO
Labeled
rhEPO
Figure 6: Comparative ion abundance
ratios for the T4 peptide at three
different time points for plasma
collection. The top row is the measured
ion abundance for the unlabeled peptide
and the bottom row is the response from
the labeled peptide.
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